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Preparation of mesothelin chimeric antigen receptor modified T cells and application of T cells in pancreatic cancer treatment

A chimeric antigen receptor, pancreatic cancer technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, etc.

Inactive Publication Date: 2017-03-29
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many problems in the application of CAR-T cells in solid tumors. The differences in the characteristics of solid tumors and hematological tumors determine that this treatment model must reconsider the characteristics of cell therapy itself, the law of action and other factors.

Method used

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  • Preparation of mesothelin chimeric antigen receptor modified T cells and application of T cells in pancreatic cancer treatment
  • Preparation of mesothelin chimeric antigen receptor modified T cells and application of T cells in pancreatic cancer treatment
  • Preparation of mesothelin chimeric antigen receptor modified T cells and application of T cells in pancreatic cancer treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Insert the fusion gene fragment (GM-CSF)-scFv(meso)-CD8-CD28-CD137-CD3ζ into the lentiviral expression vector pLent-C-GFP.

[0050] Anti-meso's CAR module comments figure 1 (See appendix SEQ ID NO.1 for the complete nucleic acid sequence).

[0051] Anti-meso's CAR module sequence

[0052] (1) GM-CSF signal peptide nucleic acid artificial sequence (SEQ ID NO.2)

[0053] (2) Anti-mesothelin monoclonal antibody single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.3)

[0054] (3) CD8αHinge region nucleic acid artificial sequence (SEQ ID NO.4)

[0055] (4) CD28 transmembrane region and intracellular region nucleic acid artificial sequence (SEQ ID NO.5)

[0056] (5) CD137 intracellular nucleic acid artificial sequence (SEQ ID NO.6)

[0057] (6) CD3ζ intracellular region nucleic acid artificial sequence (SEQ ID NO.7)

[0058] According to artificial nucleic acid sequence of signal peptide, artificial nucleic acid sequence of Anti-meso, art...

Embodiment 2

[0059] Example 2: Preparation of Chimeric Antigen Receptor (GM-CSF)-scFv(meso)-CD8-CD28-CD137-CD3ζ Modified T Cells

[0060] (1) Preparation of heterogeneous T cells—CIK

[0061] Take 75ml of autologous peripheral blood from the patient, and separate peripheral blood mononuclear cells with TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biology). After inducing culture for 24 hours with a culture medium (purchased from CORNING Company, 88-551-CM) containing 1000 IU / ml of recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical), 1000 IU / ml of recombinant interleukin 2 ( (purchased from Shenyang Sansheng Pharmaceutical), 50ng / ml of OKT-3 and 5% of the patient's autologous plasma were induced and continued to be cultured for 24 hours. Doubling liquid was added every three days, cultured until the 14th day, and the positive expression rate of CD3+ and CD56+ in CIK cells was detected by flow cytometry (CD3-FITC, CD16 / CD56-PE antibodie...

Embodiment 5

[0069] Example 5: Determination of in vitro killing effect of T cell lines after genetic modification

[0070] According to different effect-to-target ratios (50:1, 25:1, 5:1, 1:1), modified cells and unmodified T cells were co-cultured with A431, A431-H9, HAY, and LDH lactate dehydrogenase was used - Cytotoxicity Assay Kit (LDH-Cytotoxicity Assay Kit, Biovision) detects the ability of genetically modified T cells to kill different types of tumor cells in vitro. The method is as follows: Target cells were plated in a 96-well plate (5×10 3 / well), set medium background, volume correction, target cell spontaneous LDH release, target cell maximum LDH release, effector cell spontaneous LDH release control well, treatment group wells, each group repeated 3 wells, the final volume of each well was the same and Not less than 100 μL. Centrifuge at 250g for 4min, and incubate at 37°C, 5% CO2 for at least 4h. 45 min before centrifugation, add 10× lysate to the target cell maximum rel...

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Abstract

The invention provides preparation of mesothelin chimeric antigen receptor modified T cells and application of the T cells in pancreatic cancer treatment, discloses a chimeric antigen receptor, particularly relates to a chimeric antigen receptor which serves as a guiding sequence by combining a granulocyte-macrophage cluster stimulating factor receptor, and further relates to the T cells expressing the chimeric antigen receptor. The T cells can kill tumor cells highly expressing mesothelin in a targeted mode and have the application of preparing drugs for treating pancreatic cancer malignant tumors and virus infective diseases.

Description

technical field [0001] The invention relates to the technical field of biology and new medicine, more specifically, it relates to the preparation of T cells modified by mesothelin chimeric antigen receptor and its application in the treatment of pancreatic cancer. Background technique [0002] Pancreatic carcinoma (pancreatic carcinoma) is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. About 90% of pancreatic cancers are ductal adenocarcinomas originating from the ductal epithelium. The early diagnosis rate of pancreatic cancer is low, and the cure rate is extremely low. It is one of the malignant tumors with the worst prognosis, and the 5-year survival rate is <1%. The main cause of death is metastatic disease caused by pancreatic cancer. Tumor invasion and metastasis are a continuous process mediated by adhesion molecules, extracellular matrix (ECM) degradation and angiogenesis by proteases. Pancreatic cancer can occur in the h...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K16/28A61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K14/7153C07K16/303C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0646C12N15/86C12N2510/00C12N2740/14043C12N2800/107
Inventor 刘明录吴东颖万磊金海峰冯健海强邦明马洪华
Owner SHANDONG XINRUI BIOTECH CO LTD
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